The invention relates to laboratory ware for chemical and biological testing and experimentation and, more specifically, to a surface that will immobilize DNA.
Polymers have been widely utilized as vessels for cell culture, biological testing, medical research and other uses. Certain polymers such as styrenic polymers and more specifically, polystyrene, are optimal for use as substrates in specialty products for biological testing, and are typically employed. Polystyrene, for example, is cheap, optically clear, and may be processed at low temperatures.
Direct immobilization of DNA to a substrate surface has many applications to a variety of assay systems in molecular biology, such as immuno-PCR or detection of nucleic acids with specifically labeled probes, i.e. target capture hybridization. Target capture hybridization relies on immobilizing capture oligonucleotide molecules (usually single strand DNA or RNA fragments that may be isolated from a natural source, or produced by standard molecular biological techniques, having from about two to forty nucleotides) on a support surface and then hybridizing labeled complementary target DNA strands to the immobilized molecules.
An aminated nucleic acid molecule is said to be xe2x80x9cimmobilizedxe2x80x9d to a solid support if it is either adsorbed to the support, or covalently bonded thereto, with sufficient strength that it cannot be removed from the support by washing with water or an aqueous buffer. A certain release of capture oligonucleotides invariably occurs with systems employing adsorption as the method of attachment. Therefore, covalent bonding of the oligonucleotide is preferred.
One method of immobilizing aminated DNA by covalent bonding involves attaching dialdehyde starch (DAS) or N-oxysuccinimide esters (NOS) to a polymer surface. In surfaces having a DAS coating, the NH2 that is attached to the aminated capture oligonucleotide covalently links to an aldehyde group from the DAS, thereby immobilizing the oligonucleotide molecule to the polystyrene surface via the coating. A method of attaching DAS to the surface of a cell culture plate is disclosed in U.S. Pat. Nos. 5,563,215 and 5,281,660.
With a NOS coating, DNA with primary amines added synthetically or by in-vitro manipulation can be directly coupled to the NOS surface. When the primary amines react with NOS groups at a slightly alkaline pH, the ester undergoes nucleophilic substitution as the amine attacks the carbonyl group and displaces the N-oxysuccinimide group. The specificity of this reaction creates the immobilization effect.
Another technique for immobilizing DNA to a substrate surface is by covalently grafting secondary amines to a polystyrene surface. In this technique, the oligonucleotide is first phosphorylated at the 5xe2x80x2 end with polynucleotide kinase and ATP. The phospholated DNA is then coupled to the secondary amines on the surface using carbodiimide for 5 hours at 50xc2x0. The DNA is thereby immobilized by a phosphoramidate bond.
A problem exists with surface treatment involving coatings such as DAS or NOS, and with attaching secondary amines. With time, or when subjected to various environmental stresses, the surface becomes unstable and the immobilization characteristics of the attached coating are diminished. Further, the coupling efficiency of binding aminated DNA to such treated plates is quite low, less than 0.1%. Also, the surface treatment of the polymer substrate adds an additional step to the manufacturing process and adds cost to the final product.
The present invention provides a method of using a product that is made of relatively pure polystyrene that covalently binds aminated DNA. The product works as well as substrates that have been treated with DAS or NOS, but without the cost of adding a coating. The present invention can also be subjected to a variety of environmental stresses without losing any of its immobilizing characteristics. Additionally, since the product of present invention is reactive toward amine groups in general, it is capable of binding peptides and low molecular weight antigens for immunoassays, as well as antibodies, cell attachment factors and other bioreactive molecules.
The present invention provides a method of immobilizing aminated DNA to a substrate surface. The surface can be widely employed in a variety of molded laboratory products including multiwell plates, cell culture dishes, and biological containers. The method comprises the steps of providing a polystyrene substrate having a surface content of between 0.3 and 4.0 atomic percent oxygen, and attaching amine modified oligonucleotide to the surface of the substrate. A resultant product comprises a polystyrene substrate having a surface oxygen content of between 0.3 and 4.0 atomic percent, and a plurality of amine modified oligonucleotides directly attached to the surface of the substrate.